Bacterial DNA polymerases participate in oligonucleotide recombination

Abstract

SummarySynthetic single-strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have yet to be determined. Towards this end, we have identified Escherichia coli functions that process the recombining oligo and affect bacteriophage l Red-mediated oligo recombination. To determine the nature of oligo processing during recombination, each oligo contained multiple nucleotide changes: a single base change allowing recombinant selection, and silent changes serving as genetic markers to determine the extent of oligo processing during the recombination. Such oligos were often not incorporated into the host chromosome intact; many were partially degraded in the process of recombination. The position and number of these silent nucleotide changes within the oligo strongly affect both oligo processing and recombination frequency. Exonucleases, especially those associated with DNA Polymerases I and III, affect inheritance of the silent nucleotide changes in the oligos. We demonstrate for the first time that the major DNA polymerases (Pol I and Pol III) and DNA ligase are directly involved with oligo recombination.

Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

“…For instance, during ssDNA recombination, mutations in DNA replication proteins Pol I, Pol III, and DNA primase alter the frequency and/or final product of recombination (67, 72, 101). Current models for ssDNA recombination suggest that a β-coated ss-oligo is annealed at the DNA replication fork (Figure 3 a ) and that this occurs most readily when the oligo can anneal on the lagging-strand template (20, 30, 47).…”

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

“…Additionally, if the end homology is sufficient for beta‐mediated annealing (Fig A), the position of mutations within the target should not impact recombination efficiency (Li et al , ). While the mutation frequency in the donor was consistently high, we observed a decrease in the mutation frequency per residue with increasing distance from PAM on the genome (Figs D and EV1C).…”

“…Replication forks with beta‐annealed ssDNA are usually resolved by native DNA polymerases and ligases (Sawitzke et al , ; Li et al , ; Fig A). Mismatches between the wild‐type sequences and the recombination substrates are corrected by methyl‐directed mismatch repair (MMR) to reduce recombination efficiency (Costantino & Court, ; Sawitzke et al , ).…”

DNA gap repair in Escherichia coli for multiplex site‐directed mutagenesis

“…In recombineering DNA integration, defining “hArms” is challenging, with the exception of ssODNs with long heterological sequences. For example, two complementary ssODNs with a mismatch in every third base cannot be annealed, but two complementary ssODNs with a mismatch in every sixth base can be annealed 15 . In other words, sequences with short interruptions of homology are still considered hArms.…”